EHD1促进完整自噬机制及其在非小细胞肺癌顺铂耐药中的作用

Cisplatin is the core of chemotherapy regimens for non-small cell lung cancer (NSCLC). But the effect is seriously limited by the occurrence of cisplatin-resistance, of which the mechanism is remaining unclear. Recently, autophagy induced by cisplatin has been observed contributing to chemo-resistance, while only dismal results came from clinical trial involving autophagy inhibitors in cancer treatment. We reported, for the first time, high level of EHD1 is correlated with cisplatin-resistance in NSCLC. However, the mechanism under it remains unclear. In our pilot study, it’s observed that cisplatin treatment enhances EHD1-SNAP29 interaction in cisplatin-resistant NSCLC cells, while knocking-down EHD1 hindered autophagosome-lysosome fusion and rescued cisplatin-resistance. Therefore, we postulated that the interaction between EHD1 and SNAP29 facilitates cisplatin-induced autophagy progression, which is responsible for cisplatin-resistance. We are planning to verify .EHD1 being involved in cisplatin-resistance, to clarify EHD1-SNAP29 interaction being critical to autophagosome-lysosome fusion in vitro; to explore the effect of knockdown EHD1 or autophagy inhibitors on rescuing cisplatin-resistance in vivo. The results of this study could provide valuable information helpful to clarify the mechanism of cisplatin-resistance, and could put new sights into precisely selecting patients suitable to autophagy inhibitors overcoming cisplatin-resistance.

顺铂是非小细胞肺癌（NSCLC）化疗的核心药物，但顺铂耐药严重制约NSCLC的化疗效果，其机制尚未完全阐明。最近研究发现，顺铂诱导的自噬参与了耐药过程，然而在临床试验中自噬抑制剂并未凸显疗效。我们首次在NSCLC中观察到EHD1表达促进顺铂耐药，而EHD1参与顺铂耐药的机制国内外尚无研究。我们前期研究证实，顺铂耐药NSCLC细胞中存在EHD1-SNAP29相互作用，EHD1表达可增强自噬及诱导耐药，敲除EHD1可以阻止自噬体和溶酶体融合并逆转其耐药性。据此我们推测EHD1-SNAP29相互作用促进顺铂诱导的自噬完成，进而参与自噬相关耐药。本研究拟在体外研究证实EHD1-SNAP29结合是自噬溶酶体生成的关键环节，证明此机制参与顺铂耐药；拟在实验动物中明确EHD1敲除或自噬抑制剂处理对顺铂耐药逆转的作用。本研究结果对阐明顺铂耐药机制有重要意义，也为应用自噬抑制剂逆转顺铂耐药提供精准标志物。

本研究在原有研究基础上，首先明确了在NSCLC患者的癌组织中EHD1表达明显高于正常肺组织，与低EHD1表达患者相比，高EHD1 表达的患者LNR显著增高，临床进展期和EHD1的高表达是较差OS和DFS的独立预测因子。EHD1与肿瘤细胞的侵袭与转移呈正相关，并且是非小细胞肺癌不良预后的独立预测因。基因芯片结果显示EHD1与多个蛋白表达相关，其中包括自噬相关蛋白。.在体外试验中，我们选用了非小细胞肺癌细胞株A549，增加EHD1的表达，可促进DDP诱导的自噬。而降低EHD1的表达，可减少DDP诱导的自噬。在进一步的研究中，我们发现，EHD1通过与SNAP29的相互作用，参与自噬溶酶体的形成，影响完整的自噬过程。删除EHD1蛋白中EH结构域所在的羧基末端氨基酸，可抑制EHD1对自噬的增强作用。选用的HeLa细胞的平行实验也得到类似结果。.以上问题的解决将能够验证我们提出的“EHD1可以通过影响自噬参与肿瘤化疗耐药”的假说。本课题的完成，为肿瘤防治防治提供一种新思路和新靶点，具有重要的理论意义和应用价值。